Pertussis, a localized respiratory infection by B. pertussis, is a public health problem, with the number of cases in the US reaching levels not seen since the 1960's, despite recent addition of a booster for adolescents and adults. Studies of pertussis re-emergence have yielded concern about duration of protection by acellular vaccines and have led to discussion of the next generation of pertussis vaccines, as well as unanswered questions about pertussis pathogenesis. Adenylate cyclase toxin (ACT) is a B. pertussis virulence factor, which produces cAMP from host ATP, oligomerizes to form transmembrane pores and causes cytotoxicity by ATP depletion. It acts locally in the respiratory tract and is key in resisting bacterial clearance by neutrophils (PMN), yet its effects on the respiratory epithelium have not been studied. We will examine, by parallel in vitro approaches, the effects of ACT on two cell types involved in early infection, respiratory epithelial cells and PMN. Wild type (WT) and mutant forms of ACT will be tested for their effects on these cell types and WT and mutant strains of B. pertussis will be studied using in vitro infections of the same cells. The characterization of ACT effects will include kinetics and concentration dependence of toxin production and action. Since ACT does not act in isolation during infection, we will examine specific scenarios in which ACT effects overlap or conflict with those of other factors, pertussis toxin, filamentous hemagglutinin and tracheal cytotoxin. We will in SA1, study the effects of ACT on: (1A) the viability of human PMN, distinguishing among the known pathways leading to PMN death; (1B) phagocytosis, generation of reactive-oxygen species, NET formation and bacterial killing by PMN; and (1C) the production of cytokines/chemokines by PMN in response to B. pertussis and other stimuli. In SA2, we will investigate effects of ACT and Bordetella organisms on functions of the respiratory epithelium: (2A) the alteration of barrier function; (2B) cytokine/chemokine production and release; (2C) mucus production and release; and (2D) alterations in the functions of human ciliated cells in primary culture. Finally, in SA3, we will investigate effects of ACT, alone and from B. pertussis on the interactions between PMN and the respiratory epithelium, using data from SA1 and SA2. We will study the following functions, before, during and after PMN transmigration: barrier function and morphology of the epithelial monolayer, viability of the PMN and epithelial cells; bacterial killing by the PMN; cytokine/chemokine production and release by PMN and epithelial cells; and ciliary function and mucus production. At the completion of this project, we will have identified and characterized ACT effects on human PMN and airway epithelial cells, providing new perspectives on pertussis pathogenesis and critical information for consideration of ACT as an antigen in the next generation acellular pertussis vaccine.